Enzymatic proteins are remarkable natural catalysts in that they selectively perform a myriad of reactions under relatively mild conditions. Enzymes also offer the possibility of stereo-, regio- and other selectivities for the formation of novel chemicals for drugs, food additives, agricultural chemicals and other products.
Although the use of enzymes is commonly associated with aqueous chemistry, recent work demonstrates that enzymes are both active and resilient in organic media, allowing a far greater range of substrates to be examined in conjunction with a particular protein. If the substrate is insoluble or only slightly soluble in water, the maximum activity of the enzyme cannot be achieved in an aqueous solution. The study of enzyme activity in non-aqueous media is thus motivated principally by the need to extend the applicability of enzymes to the catalysis of reactions whose reactants and/or products are not water-soluble, as are many medically and industrially important reactions.
Other factors also make non-aqueous enzymology attractive. For example, the absence of water in some cases can prevent undesirable side reactions. It has also been shown that reduced water content improves the thermostability of many enzymes.
Many enzymes can function efficiently, maintaining their structure, mechanism, specificity, and stability, when suspended as lyophilized powders in a variety of anhydrous organic solvents. For enzyme reactions which do not utilize water as a reactant, such as alcohol dehydrogenases or dehalogenases, the specific activity of the enzyme can rival those solubilized in more conventional surroundings. The dependence of enzyme structure and function on solvent physical properties is currently under intensive study in many laboratories around the world. However, for those enzymes which require water as a reactant, a compromise must be found in which the enzyme has access to essential water, while the substrate can be solubilized in the organic solvent of choice.
The use of biphasic mixtures of aqueous and organic phases has been suggested by several researchers. In these systems, the enzyme is contained within the aqueous phase and the substrate is introduced in the organic phase. While this approach enables a higher substrate concentration as well as offering a convenient method for product removal via the organic phase, several disadvantages are associated with it. Because of the low solubility of the substrates in the aqueous phase where the enzyme is located, very high concentrations of the substrate (on the order of moles/liter) are necessary to enhance the product formation. The use of very low volumes of the water phase can achieve the same result. The use of these systems for preparative chemistry has been described.
Another route to achieving some of the advantages of non-aqueous enzymology while maintaining the aqueous environment around the enzyme is the use of reversed micelles, which are spherical aggregates of amphiphilic molecules in an organic solvent encapsulating a water pool.
A further recent trend in biotechnology involves the use of modified enzymes which are soluble and active in organic solvents. Recent work has shown that one can covalently functionalize an enzyme with an amphiphilic polymer such as polyethylene glycol (PEG), thus allowing the enzyme adduct to dissolve in an organic solvent while maintaining activity. The covalent attachment can be accomplished by linkage of the .epsilon.-amino groups of lysine residues to derivatives of PEG. The resulting PEG-modified enzymes are soluble to concentrations of a few mg/ml in a number of organic solvents. The homogenous PEG-enzyme system resulting in enzyme (protein) solubilization is not limited by diffusion, and enables the enzyme to retain high activity.
Most of the recent studies of enzyme behavior in organic media employ conventional liquid solvents, yet some new and intriguing applications may become possible if one could tether an enzyme to an organic polymer. Indeed, the covalent attachment of proteins, including enzymes, to polymers has been the subject of intensive research for many decades. Immobilized proteins have been used in the food, chemical, pharmaceutical, and agricultural industries. Nevertheless, the variety of polymers which can serve as suitable hosts for biological activity are limited by the conditions under which proteins are typically incorporated into polymers. The predominant reason for this apparent limitation is related to the sensitivity of most proteins to their environment. For instance, a protein which is unstable at pH 9.0 is unlikely to be successfully immobilized using a strategy which is dependent on alkaline pH's during attachment.
Further, the development of a wide variety of classes of protein-containing polymers depends not only on the availability of immobilization strategies, but also on the stability of proteins in environments which are best suited to rational polymer design. The potential application of biomaterials would be enhanced by redirecting the focus on choosing a given set of polymer properties, and having a general method for incorporating a protein in the favored polymer matrix.
It is an object of the present invention to provide a general method for synthesizing novel protein-containing polymers in which biological activity is retained in both aqueous and non-aqueous environments as well as mixtures thereof.